Microbes have begun to be developed for use as vaccines that deliver heterologous antigens. Heterologous antigen delivery is provided by microbes that have been modified to contain nucleic acid sequences encoding a protein or antigen originating from a different species. Heterologous antigen delivery is especially advantageous for treating or preventing diseases or conditions that result from especially virulent or lethal sources, such as cancer and pathogenic agents (for example, HIV or Hepatitis B), wherein injection of a native infectious agent or cancer cell is potentially deleterious to the recipient organism, and administration of attenuated or killed agent or cell has proven unsuccessful in eliciting an effective immune response, or where sufficient attenuation of the infectious agent or cancer cell cannot be assured with acceptable certainty. Recently, certain bacterial strains have been developed as recombinant vaccines. For instance, an oral vaccine of attenuated Salmonella modified to express Plasmodium berghei circumsporozite antigen has been shown to protect mice against malaria (Aggarwal et al. 1990. J. Exp. Med. 172:1083).
Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular bacterium that is being developed for use in antigen-specific vaccines due to its ability to prime a potent CD4+/CD8+ T-cell mediated response via both MHC class I and class II antigen presentation pathways. See, for instance, U.S. Pat. Nos. 6,051,237, 6,565,852, and 5,830,702.
Listeria has been studied for a number of years as a model for stimulating both innate and adaptive T cell-dependent antibacterial immunity. The ability of Listeria to effectively stimulate cellular immunity is based on its intracellular lifecycle. Upon infecting the host, the bacterium is rapidly taken up by phagocytes including macrophages and dendritic cells (DC) into a phagolysosomal compartment. The majority of the bacteria are subsequently degraded. Peptides resulting from proteolytic degradation of pathogens within phagosomes of infected APCs are loaded directly onto MHC class II molecules, and the processed antigens are expressed on the surface of the antigen presenting cell via the class II endosomal pathway, and these MHC II-peptide complexes activate CD4+ “helper” T cells that stimulate the production of antibodies. Within the acidic compartment, certain bacterial genes are activated including the cholesterol-dependent cytolysin, LLO, which can degrade the phagolysosome, releasing the bacterium into the cytosolic compartment of the host cell, where the surviving Listeria propagate. Efficient presentation of heterologous antigens via the MHC class I pathway requires de novo endogenous protein expression by Listeria. Within the cytoplasm of antigen presenting cells (APC), proteins synthesized and secreted by Listeria are sampled and degraded by the proteosome. The resulting peptides are shuttled into the endoplasmic reticulum by TAP proteins and loaded onto MHC class I molecules. The MHC I-peptide complex is delivered to the cell surface, which in combination with sufficient co-stimulation (signal 2) activates and stimulates cytotoxic T lymphocytes (CTLs) having the cognate T cell receptor to expand and subsequently recognize the MHC I-peptide complex displayed on, for example tumor cells. In the appropriate microenvironment, the activated T cell targets and kills the cancerous cell.
Given the mechanisms by which Listeria programs the presentation of heterologous antigens via the MHC class I pathway, the efficiency of both expression of heterologous genes and secretion of the newly synthesized protein from the bacterium into the cytoplasm of the infected (antigen presenting) cell is directly related to the potency of CD8+ T cell priming and/or activation. Since the level of Ag-specific T cell priming is directly related to vaccine efficacy, the efficiency of heterologous protein expression and secretion is linked directly to vaccine potency.
Thus, novel methods are needed in the art to optimize the efficiency of heterologous protein expression and secretion to maximize the potency of Listeria-based vaccines and other bacteria-based vaccines. It would also be beneficial to optimize the efficiency of heterologous protein expression and secretion in bacterial host expression systems where expression and secretion of large quantities of heterologous protein is desired.